Transmitter release from vertebrate hair cells occurs at ribbon synapses where glutamate-containing vesicles fuse with the plasma membrane upon a rise in cytoplasmic calcium. Voltage-sensitive calcium channels are gated by receptor potentials to cause transmitter release that then triggers action potentials. Remarkably, in the mammalian cochlea each afferent neuron makes a single postsynaptic contact with an inner hair cell, where usually a single ribbon provides the requisite glutamate release. Thus, throughout the lifetime of the organism, a single ribbon synapse is responsible for signaling all that neuron can report to the brain concerning the frequency, timing and intensity of sound, including spontaneous activity as high as 100 Hz in the absence of sound! How does an individual ribbon carry out this task? Recent synaptic recordings from single afferent dendrites have suggested that the gating of single VGCCs is critical for information transfer at the ribbon synapse. Hair cell VGCCs are dihydropyridine-sensitive, rapidly gating at relatively negative membrane potentials, and show little inactivation in most studies. There is good evidence that the majority of VGCCs in mammalian inner hair cells contain the Cav1.3 (a1D) pore-forming subunit. However, heterologous expression of that gene product results in channels whose properties differ variously from those of the native hair cell VGCCs. Most notably, the cloned channel shows robust calmodulin-dependent calcium inactivation. Several possibilities exist to explain this difference, including alternative splicing of Cav1.3 mRNA in hair cells, and combination with various modulatory proteins. In this proposal we will combine the expertise of three laboratories to examine these possibilities. Dr. Tuck Wah Soong will use his technique of exon-scanning to characterize alternative splicing in cochlear hair cells. Dr. David Yue will apply his knowledge to develop further hypotheses of calmodulin and calmodulin-like protein modulation of hair cell VGCCs. The Fuchs laboratory will complete basic descriptions of GDI in cochlear hair cells, provide materials for gene-amplification by Soong, and together with Yue and Soong, employ new molecular tools in to alter native VGCCs in hair cells. The steady-state and dynamic gating of VGCCs will be determined by the balance of calcium-dependent inactivation and opposing mechanisms. Our ultimate aim will be to derive the contribution of VGCC gating to hair cell information transfer. [unreadable] [unreadable] [unreadable]